Fruit quality by inhibiting production of lipoxygenase in fruits

ABSTRACT

The present invention relates generally to a transgenic fruit-bearing plant having a foreign nucleotide sequence inserted into its genome which is substantially similar to a portion of the plant&#39;s fruit ripening specific lipoxygenase cDNA. Transgenic plants according to the present invention produce fruits having modified and surprisingly superior ripening characteristics, including improved quality and texture, greater firmness, longer shelf life, better packaging and storage characteristics, and improved processing characteristics. Also provided are transgenic fruits; transgenic plant cells; methods for making transgenic plants, fruits and plant cells; methods for inhibiting lipoxygenase production in plants; isolated nulceic acid sequences; and vectors comprising these isolated nucleotide sequences.

This application is a 371 of PCT/US96/16387 filed Oct. 11, 1996, whichclaims priority to provisional application Number 60/005,404 filed Oct.13, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated nucleotide sequences; methodsfor using the sequences to make transgenic plants which produce ediblefruits having improved firmness and longevity; and vectors having thenucleotide sequences incorporated therein. More specifically, thepresent invention relates to transgenic plants and methods for makingthe same, the genomes of said plants having incorporated therein foreignnucleotide sequences which function to inhibit production of fruitripening specific lipoxygenase (“FRS-LOX”) in a ripening fruit. Theinhibition of FRS-LOX gene expression provides a mechanism to improvecharacteristics of a fruit, including controlling fruit senescence andtissue softening associated with post-maturation Processes.

2. Discussion of Related Art

Biochemistry of Lipoxygenases

Lipoxygenases (“LOXs”) are nonheme iron-containing dioxygenases thatcatalyze the incorporation of molecular oxygen into unsaturated fattyacids containing cis, cis 1,4-pentadiene structure to yield a1-hydroperoxy-2-4-trans, cis pentadiene product. Typical substrates forLOXs in plants are linoleate (C18:2) and linolenate (C18:3) fatty acids,while animal LOXs prefer arachidonate (C20:4). Some LOXs are able to acton substituted fatty acid substrates, while others require free fattyacids. LOXs can vary in respect to (1) the site of primary hydrogenabstraction, (2) the direction of the double bond shifts in the primaryradicle leading to the hydroperoxide product and (3) thestereospecificity of both hydrogen abstraction and dioxygen insertion(Ford-Hutchinson et al., Annu. Rev. Biochem. 63:383, 1994).

In plants, most fatty acids are esterified to a glycerol backbone in theform of glycerolipids and lipases are thought to cleave thesephosopholipids into usable plant LOX substrates (free fatty acids). TheLOX-catalyzed fatty acid hydroperoxides serve as intermediates for anumber of secondary reactions leading to jasmonic acid, traumatin,traumatic acid, volatile alcohols (hexanal), aldehydes, ketols and 9Coxo fatty acids (Vick and Zimmerman, Biochemistry of Plants, Vol. 9:53,1987; Hildebrand, Physiol. Plantarum 76:249, 1989). The 5-LOX fromhumans is representative of a unique type of LOX that requires theassociation of the 5-LOX Activating Protein (FLAP) for activity. Also, aLOX from the rabbit reticulocyte has been reported to attackmitochondrial membranes in the absence of any lipid hydrolyzing enzymesduring the maturation of erythroid cells (Schewe et al., Adv. Enzymol.58:191, 1986).

Lipoxygenases: A Multigenic Family

LOXs have been found in a wide range of organisms including higherplants, animals, yeast, fungi and cyanobacterium (Siedow, Annu. Rev.Plant Physiol. Plant Mol. Biol. 42:145, 1991). LOX multigenic familieshave been characterized in several plant and animal species (Siedow,Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:145, 1991; Ford-Hutchinsonet al., Annu. Rev. Biochem. 63:383, 1994). The best characterized plantLOXs are the three soybean cotyledon LOX monomer isozymes; L-1, L-2 andL-3, all globular, water soluble proteins with MWs of about 96 kD. LOXsfrom rice, soybean, cotton, sunflower, tomato, Arabidopsis, cucumber,kiwi and tobacco are some that have been identified and are on the orderof 95 kD, with the exception of pea (72-108 kD). Sequences reported forplant LOXs are approximately 60% homologous to one another. Human LOXsare about 60% homologous and are only 25% identical to plant LOXs(Ford-Hutchinson et al., Annu. Rev. Biochem. 63:383, 1994). Some of theplant LOXs are larger than animal LOXs and show homology with them onlyin limited regions (Minor et al., Biochem. 32:6320, 1993).

Biological Role(s) of Lipoxygenases

The function of various LOX isozymes in plant and mammalian systems isunknown. The hydroperoxide products from some animal LOXs serve asprecursors in the production of leukotrienes and lipoxins, regulatorymolecules involved in responses include leukotiene induced altered cellfunctions such as chemotaxis and chemokinesis. Roles of LOX during allstages of plant growth and development have been speculated (Siedow,Annu. Rev. Plant Physiol. Plant Mol. Bio. 42:145, 1991). LOX activityhas been demonstrated in rapidly growing young tissues (germinatingseedlings). It has been suggested that jasmonic acid and hydroperoxidefree radicles, primary and secondary products of LOX, may play roles inplant senescence by promoting cell membrane deterioration, inhibitingprotein synthesis and chloroplast photochemical activity. Upon tissuewounding, increases in LOX activity and mRNA accumulation have been seenin some plants. Traumatin and traumatic acid may be involved in woundhealing of damaged plant tissues (Hildebrand, Physio. Plantarum 76:249,1989). Another secondary LOX product, hexanal, may be produced inresponse to pest/pathogen attack. However, no evidence to support any ofthese hypotheses has been obtained.

Shelf Life of Fruits and Vegetables

Fruits and vegetables are highly perishable crops and significant lossesoften occur after their harvest but before they reach the consumer. Theprimary causes of these losses are the inability to control: 1)senescence of these crops; 2) the ripening process in fruits; and 3)ripening-and sensescence-associated tissue softening. One of theobjectives of plant breeders for many years has been to control theseprocesses by introducing traits from wild germ plasm into the cultivatedspecies. In recent years, attempts have been made to use recombinant DNAtechnology to modify some of these traits. Both antisense andco-suppression technologies have been used in some crop plants to modifythe expression of specific genes which may have deleterious effects onplant growth and development or crop productivity in general. However,none have proven fully satisfactory in reducing the rate of tissuesoftening associated with post-maturation plant processes.

Major transitions in fruit development and metabolism accompany theinitiation of fruit ripening. In addition to alterations in pigmentbiosynthesis and production of volatile compounds, fruits undergosignificant changes in texture during ripening. The biochemical basesfor ripening- and senescence-associated fruit softening are not yetunderstood; however, dissolution of the middle lamella and cell wallseparation due to depolymerization of pectins by polygalacturonase aswell as loss of calcium have been suggested to contribute to fruitsoftening. Only slight improvement in fruit integrity has been reportedin fruits with low polygalacturonase activity (Schuch et al. HortScience26: 1517, 1991; Carrington et al., Plant Physiology 103: 429, 1993).

There is a need for transgenic plants which produce fruits havingmodified ripening and post-maturation characteristics including improvedquality and texture, greater firmness, longer shelf life, better packingand storage characteristics and improved processing characteristics.

SUMMARY OF THE INVENTION

The invention described herein features inhibiting the expression offruit ripening specific lipoxygenase (“FRS-LOX”) in a plant,specifically in cells of a fruit of the plant. For instance, foreign DNAcan be introduced into cells to reduce FRS-LOX production. In apreferred embodiment, the cell is a plant cell and the genetic materialis a sense or antisense fragment substantially similar to a portion ofthe FRS-LOX cDNA shown in SEQ ID NO:4. Most preferably, the cell is acell of a fruit-bearing plant, such as a tomato plant cell.

It is expected that the present invention can be applied to theinhibition of FRS-LOX gene products in a wide variety of useful plants.These may include, for example, commercially important fruit-bearingplants in which post-maturation weakening reduces economic value, suchas melons, peaches, bananas, apples, strawberries, kiwi fruit, and inparticular the tomato.

The present inventors have made the surprising discoveries that (1)inhibition of FRS-LOX greatly improves fruit qualities such as, forexample, firmness and shelf life; and (2) antisense and co-suppression(sense) technologies can be successfully used to inhibit FRS-LOX geneexpression in plants so as to provide fruits having superior qualitiessuch as, for example, firmness and shelf life. It is believed thatimproved fruit qualities result from reduced activity of degradativepathways (e.g., membrane deterioration); however, it is not intendedthat the present invention be limited to this theory.

Briefly describing one aspect of the present invention, there isprovided a method for making a transformed plant having improved fruitquality comprising inserting a nucleotide sequence into DNA of the plantin a sense or antisense orientation under a suitable promoter so as toinhibit production of lipoxygenase in the fruit of the plant as itripens. In a preferred method, a sequence of nucleotides is insertedinto a target cell by providing a vector comprising the nucleotidesequence and contacting the vector with the target cell.

Additional aspects of the present invention include vectors havingincorporated therein nucleotide sequences having substantial similarityto all or a portion of the sequence of SEQ ID NO:4(FIG. 4).

Additional aspects of the invention include constructs selected from thegroup consisting of pMLSL, PMLAL, pUSL2, pUAL2, pUEL300S and pUEL300A,which are useful for inserting foreign DNA into a plant cell genome.

According to other aspects of the present invention there are providedtransgenic fruits, transgenic plants and transgenic host cells,preferably plant cells such as germ cells and cotyledon cells. Thesevarious transgenic hosts are preferably transformed by havingincorporated into their genomes, nucleotide sequences as delineatedabove and described in greater detail below.

It is an object of the present invention to provide transgenic plantswhich produce fruits having modified ripening characteristics, includingimproved quality and texture, greater firmness, longer shelf life,better packaging and storage characteristics and improved processingcharacteristics.

Further objects, advantages, and features of the present invention willbe apparent from the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the nucleotide sequence of one portion of the fruitripening specific lipoxygenase (“FRS-LOX”) gene which is inserted into aplant cell genome according to one preferred embodiment of the presentinvention.

FIG. 2 sets forth the nucleotide sequence of another portion of theFRS-LOX gene which is inserted into a plant cell genome according toanother preferred embodiment of the present invention.

FIG. 3 sets forth the nucleotide sequence of another portion of theFRS-LOX gene which is inserted into a plant cell genome according toanother preferred embodiment of the present invention.

FIG. 4 sets forth the nucleotide sequence of the coding region of atomato FRS-LOX gene cloned and characterized for use in accordance withthe present invention.

FIG. 5 shows various regions of the tomato FRS-LOX gene used for makingvectors according to the present invention.

FIG. 6 is a plot of fruit firmness vs. the number of days after breakerfor a transgenic tomato of the present invention (650-1) and an Ohiocontrol tomato. “Breaker,” as used herein, is intended to denote thetime at which a tomato begins to change color from green to orange.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, references will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended. Any alterations and modifications in theinvention, and such further applications of the principles of theinvention therein being contemplated as would normally occur to oneskilled in the art to which the invention relates.

In accordance with the present invention, there are provided transgenicplants and fruits and methods and materials for making them. It has beendiscovered that the expression of the fruit ripening specificlipoxygenase (“FRS-LOX”) gene, and correspondingly, the level of FRS-LOXenzyme activity, are implicated in causing fruit weakening and membranedeterioration in post-maturation fruits. It has also been discoveredthat these processes can be suppressed in plants by the introduction ofa foreign nucleotide sequence having substantial similarity to all or aportion of the coding sequence of the FRS-LOX gene (e.g., as set forthin FIG. 4, SEQ ID NO:4) in either the sense or the antisenseorientation. The invention thus provides recombinant DNA with which toachieve such suppression, methods for transforming plants to achievesuch suppression, and the resultant transgenic plant cells, transgenicplants and transgenic fruits thereof. The term “substantial similarity,”as used herein, is intended to mean sufficiently similar to causeimproved fruit quality by inhibiting lipoxygenase production in a fruitwhen inserted in sense or antisense orientation. For example, it iscontemplated that nucleotide sequences useful in the invention willhybridize, under stringent hybridization conditions, to the codingsequence of the FRS-LOX gene (e.g., the nucleotide sequence of SEQ IDNO:4; FIG. 4).

The Examples given below clearly show that the methods of the invention,using the expression of a nucleotide sequence comprising a portion ofthe FRS-LOX cDNA, results in substantial inhibition of FRS-LOX. Tomatofruit and their seeds, for example, and progeny of these plants willfind use in the production of new tomato varieties containing reducedFRS-LOX. These plants are useful in the production of tomatoes ofimproved quality, which have a longer storage life, bettertransportablility, better field holding (i.e, fruit lasts longer in goodcondition on the plant prior to harvest) or be easier to process, andmay produce improved products such as whole peeled tomatoes, puree,ketchup or sauces. It will be understood that an FRS-LOX gene appearsnot only in tomatoes, but also in a wide variety of other plants andthat the invention can be used not only for the inhibition of tomatoFRS-LOX, but also for the inhibition of FRS-LOX and similar enzymes invarious other fruit-bearing plant species. In these other species, ofcourse, the inserted foreign nucleotide sequence must have substantialsimilarity to all or a portion of the FRS-LOX gene associated with thatspecies.

The preferred aspects of the present invention are carried out using theFRS-LOX gene. The cDNA clone for a tomato FRS-LOX gene (shown in FIG. 4;SEQ ID NO:4) has 2871 nucleotide base pairs and an open reading frameencoding a protein of 859 amino acids with calculated molecular mass of97 kD and pI of 5.5. Comparison of its sequence reveals 73 and 82percent similarity at nucleic acid and amino acid levels, respectively,to a LOX cloned from wounded potato tubers which show 5-LOX activity onarachidonic acid (Mulliez et al., Biochem. Biophys. Acta 916,13, 1987;Casey, Plant Physiology 107:265, 1995).

In accordance with the present invention, a nucleotide sequence havingsubstantial similarity to all or a portion of the coding sequence of theFRS-LOX gene (SEQ ID NO:4) is incorporated in a recombinant DNA moleculeunder the control of a promoter. In this regard, a recombinant DNAmolecule is one which has either been naturally or artificially producedfrom parts derived from heterologous sources, which parts may benaturally occurring or chemically synthesized molecules, and whereinthose parts have been joined by ligation or other means known in theart. The introduced FRS-LOX coding sequence is under control of thepromoter and thus will be generally downstream from the promoter. Statedalternatively, the promoter sequence will be generally upstream (i.e.,at the 5′ end) of the coding sequence.

A constitutive promoter was used in the methods described in theExamples below. However, targeting of the gene product can be obtainedusing a constitutive (e.g. Cauliflower Mosaic Virus 35S promoter),inducible (e.g. Tomato E8 ethylene inducible promoter) ordevelopmentally regulated (e.g. Tomato polygalacturonase promoter)promoter to construct the vectors.

With respect to the function of the promoter, it is well known thatthere may or may not be other regulatory elements (e.g., enhancersequences) which cooperate with the promoter and a transcriptional startcodon to achieve transcription of the introduced (i.e., foreign)sequence. The phrase “under control of” contemplates the presence ofsuch other elements as are necessary to achieve transcription of theintroduced sequence. Such transcription can be assessed, for example, bythe detection of the mRNA products of the same. Also, the recombinantDNA will preferably include a termination sequence downstream from theintroduced sequence.

The introduced sequence according to the instant invention is preferablya nucleotide sequence having substantial similarity to all or a portionof the nucleotide sequence that encodes the FRS-LOX enzyme (see forexample, SEQ ID NO:4). As used herein, the term “portion” is intended torefer to a nucleotide sequence having a sufficient number of nucleotidesto cause improved fruit quality by inhibiting lipoxygenase production ina fruit when inserted in sense or antisense orientation. In this regard,coding sequences of about 90 base pairs have been shown to possessinhibitory properties in sense interactions. (See Vaueheret H. (1993)Identification of a General Silencer for 19S and 35S Promoter in aTransgenic Tobacco Plant: 90 bp of homology in the promoter sequence aresufficient for transactivation. C.R. Acad. Sci. III 316:1471-1483.)

According to the present invention, the introduced sequence can haveeither a sense or an antisense orientation, and can contain nucleotidesequences from any suitable source, including both natural and syntheticsources. In a preferred embodiment, the introduced sequence is one ofthose of FIGS. 1-3 (SEQ ID NOS:1-3) or one having substantial similarityto one of these sequences, and is incorporated in either a sense or anantisense orientation.

One preferred vector according to the present invention, for example thepMLSL construct, comprises the nucleotide sequence shown in FIG. 1 (SEQID NO:1) in the sense orientation (See Example 3, below). The DNA insertshown in SEQ ID NO:1 is a 2440 base pair DNA fragment representingnucleotides 158 to 2598 of the fruit FRS-LOX gene shown in FIG. 4 (SEQID NO:4). Another preferred vector, for example the pMLAL construct (SeeExample 3, below), comprises the same DNA fragment inserted into thevector in the antisense orientation (reverse order).

Another nucleotide sequence which is advantageously inserted into avector according to the present invention is a sequence containing thefull-length FRS-LOX cDNA (SEQ ID NO:4) having 2871 base pairs. Thisnucleotide sequence is shown in FIG. 2 (SEQ ID NO:2). This sequence mayalso be inserted in either the sense orientation (See, for example, thepUSL2 construct described in Example 4, below) or the antisenseorientation (See, for example, the pUAL2 construct construct describedin Example 4, below).

A third nucleotide sequence which is inserted into a vector according toa preferred embodiment of the present invention is a sequence containingnucleotides 1 through 297 of the FRS-LOX gene shown in FIG. 4 (SEQ IDNO:4). This sequence is also set forth in FIG. 3 (SEQ ID NO:3). As withthe previously-described sequences, this nucleotide sequence may beinserted into a vector in either the sense orientation (see, forexample, the pUEL300S construct described in Example 5, below) or theantisense orientation (see, for example, the pUEL300A constructdescribed in Example 5, below). These vectors are all useful for makinginventive transgenic plants as described above.

Additionally contemplated by the present invention is a vector havingincorporated therein a nucleotide sequence substantially similar to oneof the above-described :nutcleotide sequences.

Suitable nucleotide sequences for use as starting materials in thepresent invention can be isolated from DNA libraries obtained from otherspecies by means of nucleic acid hybridization or PCP, using ashybridization probes or primers for FRS-LOX, nucleotide sequences thathave been published for FRS-LOX genes. Alternatively, antibodies to theFRS-LOX protein can be used to screen a plant cDNA library for clonesthat express the FRS-LOX protein. The cDNA thus identified asFRS-LOX-protein-encoding can then be used to isolate a genomic clonecontaining a similar nucleotide sequence or a portion thereof. For anillustrative list of clones obtained in this way, see Table 1 in ExampleI below.

In accordance with the present invention, the FRS-LOX insertion sequencecan be, but is not necessarily, a mutant form. Mutations may include,for example, insertions, deletions, and/or substitutions of one or morenucleotides. Such a mutation in accordance with the invention willprovide a coding sequence which, when inserted into a plant in the senseor antisense orientation under the control of a promoter that isexpressed in the plant, achieves the suppression of the expression of anatural FRS-LOX gene and the FRS-LOX activity of the transgenic plant.

Recombinant DNA in accordance with the invention can be incorporatedinto vectors and introduced into the genome of plants using conventionaltechniques. In this regard, the term “genome” as used herein is intendedto refer to DNA which is present in the plant and which is heritable byprogeny during propagation of the plant. For example, the invention isillustrated in the Examples below utilizing Agrobacteriumtumefaciens-mediated transformation, although other techniques can alsobe used and are within the purview of the ordinarily skilled artisan.The technique used for a given plant species or specific type of planttissue will depend upon the known successful techniques. Additionalmeans for introducing recombinant DNA into plant tissue include but arenot limited to electroporation, microprojectiles, microinjection, aswell as other T-DNA mediated transfer from Agrobacterium tumefaciens.

Once the recombinant DNA is introduced into the plant tissue, successfultransformants can be screened using standard techniques such as the useof marker genes, e.g., genes encoding resistance to antibiotics.Additionally, the level of expression of the natural FRS-LOX gene oftransgenic plants may be measured at the transcriptional level or asprotein synthesized.

Transgenic plants in accordance with the present invention can also beidentified by detection of a significant increase in the firmness of thefruit of the transgenic plant as compared to its wild-type counterparts.The level of firmness of a whole fruit can be determined,for example, byusing conventional testing equipment such as a McCormick Fruit Tech(Yakima, Wash.) firmness pressure tester. This tester has a plunger tipand measures the penetration force necessary for the plunger tip toenter the fruit epidermis. In performing these tests, about a 1 cmsquare section of the waxy epidermis from each fruit was removed wherethe measurement was to be taken. A minimum of six fruits were analyzedand the mean and standard error were calculated. The results of variousfirmness tests are given in Table 3 below.

Fruits produced by transgenic plants made according to the presentinvention are superior to other fruits due to the advantageouslymodified ripening characteristics including improved quality and textureand greater firmness. These characteristics are believed to result fromreduced activity of degradative pathways in the fruit (e.g., membranedeterioration); however, the present invention is not intended to belimited by this theory. These characteristics are economicallyimportant, for example, because they impart to the fruit improved shelflife, better packaging and storage characteristics and improvedprocessing characteristics. The present invention provides fruits whichare much more easily handled than other fruits, and which are much lesslikely to be bruised or smashed in the normal course of harvesting,handling, transportation and delivery to a consumer.

According to the present invention, a partial inhibition of lipoxygenaseproduction is adequate for improving fruit quality. Varying levels ofinhibition can be obtained by either selecting other transformants,using different fragments of the nucleotide sequence of the FRS-LOX gene(e.g.,SEQ ID NO:4) or using a different promoter, as will be apparent bythose skilled in the art. In this regard, preferred transgenics of theinvention exhibit at least about a 50% reduction in the level of naturalFRS-LOX gene expression or FRS-LOX activity, and in more preferredtransgenic plants, reduction is 60% or more. Most preferably, transgenicplants of the invention exhibit at least about 70% reduction of theseprocesses.

Transgenic plants in accordance with the invention can be cultured andreproduced under standard conditions and using standard techniques.Likewise, transgenic fruits obtained from transgenic plants can beconventionally used and processed.

The invention will be further described with reference to the followingspecific Examples. However, it will be understood that the Examples areoffered to further illustrate the present invention, but are not to beconstrued as limiting the scope thereof.

EXAMPLE ONE Molecular Cloning of FRS-LOX CDNA

A 90 kd protein that accumulates in the red-ripe fruit of a tomatoplant, purified by ammonium sulfate precipitations, ion-exchangechromatography and SDS-PAGE was used to produce polyclonal antibodies ina rabbit. These antibodies were used to immunoscreen more than 150,000recombinant clones from an expression cDNA library made from poly A+RNAfrom red-ripe tomato pericarp in the UNI-ZAP XR vector. Among the 24cDNA clones obtained, 17 clones were similar and had inserts whichshowed 60-65% homology with the LOX gene family (Gen Bank and EMBLsequence libraries). One of the cDNAs obtained was used to rescreen thecDNA library to obtain a full-length FRS-LOX gene (Kausch and Handa,Plant Physiology 107:669, 1995). FIG. 4 shows the nucleic acid sequenceof this gene. Table 1 shows percent similarities of this FRS-LOX gene toLOX clones from other plant species.

TABLE 1 Comparison of the fruit ripening specific lipoxygenase genesequences at DNA and amino acid levels with other plant Lipoxygenases.Lox Gene Percent Similarity Accession # Nucleic Acid Amino Acid Leu0902599.5 99.1 Leu09026 72.7 81.7 Stloxl 73.2 81.6 Athlipoxy 64.6 75.7Athatlo 51.6 61.4 Pvlipoxy 64.8 74.9 Soylox 61.4 69.7 Soyloxb 62.8 73.3Soyloxc 62.7 67.6 Pslipox 61.4 71.9 Gmu04526 62.0 73.0 Gmu04785 54.171.2 Pslipocy 61.7 73.7 Oslma 54.1 71.2 Ric120p 48.2 61.2

EXAMPLE TWO Construction of Vectors

Several sense and antisense vectors containing different regions of thecloned FRS-LOX (FIG. 4) were made to create transgenic tomato plants.Table 2 summarizes the vectors made and specific procedures used toprepare them are described in Examples 3-6.

TABLE 2 Summary of Sense and Antisense LOX Vectors. Name of Sense Nameof Antisense Vector Vector Insert Size pMLSL pMLAL 2440 bp pUSL2 pUAL22871 bp pUEL300S pUEL 300A 297 bp

EXAMPLE THREE Construction of pMLSL/pMLAL

1. A 1.7 kb Eco RI and Cla I DNA fragment containing the CauliflowerMosaic Virus (CaMV 35S) promoter and the small subunit of the Ribulose1,2-Bisphosphate Carboxylase/Oxygenase (rbcS) 3′ terminator withintervening multi-cloning sites was isolated from pKYLX7 (Schardl etal., Gene 61:1, 1987) and cloned into pTZ18U to generate vectorpTZ35rbcS.

2. A 2440 bp DNA fragment representing nucleotides 158 to 2598 of thefruit FRS-LOX (FIG. 1, SEQ ID NO:1) was isolated form a partial LOXclone 8-27-1 and ligated into the multi-cloning sites present betweenthe CaMV 35S promoter and rbcS 3′ terminator sequences in pTZ35rbcS inboth orientations. The clone with the LOX cDNA in the sense orientationrelative to the CaMV 35S promoter was named pTZSL, while the clone withthe LOX cDNA in the antisense orientation relative to the CaMV 35Spromoter designation as pTZAL. Restriction mapping and Southern blottingwith CaMV 35S, LOX and rbcS 3′ probes and DNA sequencing were used toestablish structure of pTZSL and pTZAL.

3. The 4.1 kb DNA fragments containing the CaMV 35S promoter, 2440 bpLOX DNA fragment in sense or antisense orientation and rbcS 3′terminator were obtained from pTZSL or pTZAL, respectively. Thesefragments were cloned between the Eco RI and Sma I sites in PMLJ1 (anAgrobacterium based transformation vector) both in sense and antisenseconfiguration to obtain PMLSL and pMLAL, respectively. Restrictionmapping and Southern blotting with CaMV 35S, LOX and rbcS 3′ probes andDNA sequencing were used to establish structures of pMLSL and pMLAL.

EXAMPLE FOUR Construction of pUSL2/pUAL2

1. A 0.6 k-b DNA fragment (0.6 kb) containing the rbsS terminator wasisolated from pTZ35rbcS and cloned at Xba I site of pGEM11Z to createpG11rbcX.

2. The CaMV35S promoter (1.1 kb) was isolated from pTZ35rbcS afterdigestion with Sac I and Hind III, ligated with Hind III-Eco RI linkers,and forced cloned between Sac I and Eco RI sites of pGllrbcX to createpG35rbcX.

3. The Sac I and Hind III DNA fragment containing CaMV 35S promoter andrbcS terminator isolated from pG35rbcX was blunt ended (using T4polymerase) and ligated to the blunt ended Apa I and Sal I digestedpMLJI to create pML35rbc. This vector (8.45 kb) has been designated aspPUH11.

4. A DNA fragment containing the full-length FRS-LOX CDNA (nucleotide 1to 2871, See FIG. 2, SEQ ID NO:2) was isolated from clone 10-1#4-1 afterdigestion with Pst I, and Xho I and blunt-ended with T4 DNA Polymerase.This DNA fragment was cloned in both orientations in the blunt-endedpPUH11 at Xho I site to create pUSL2 (sense) and pUAL2 (antisense)vectors, respectively. Restriction mapping and Southern blotting withCaMV 35S, LOX and rbcS 3′ probes and DNA sequencing were used toestablish structure of pUSL2 and pUAL2.

EXAMPLE FIVE Construction of pUEL300s/pUEL300A

1. A DNA fragment containing nucleotides 1 to 297 of FRS-LOX (FIG. 3,SEQ ID NO:3) was obtained after polymerase chain reaction using aLOX-specific primer (a 17-mer oliqnucleotide with the sequence 5′GGGTGATGTCTGTAAGC3′ corresponding to the nucleotides 297-281) and the T3Primer (a 17-mer primer specific for the T3 promoter located 90 bpupstream of the FRS-LOX cDNA sequence in pBluescript). This DNA fragment(0.387 kb) was cloned into Eco RV T-tailed pBluescript KS to yieldpKSRS17.

2. The LOX specific DNA fragment was obtained from pKSRS17 after Eco RIdigestion and cloned in both orientations into an Eco RI digested pPUH11using T4 DNA ligase to create pUEL300S (sense) and pUEL300A (antisense)chimeric genes, respectively. Restriction mapping and Southern blottingwith CaMV 35S, LOX and rbcS 3′ probes, and DNA sequencing were used toestablish structure of pUEL300S and pUEL300A.

EXAMPLE SIX Transfer of Vectors to Agrobacterium

The vectors pUSL2, pUAL2, pMLSL, pMLAL, pUEL300S and pUEL300A weremobilized into Agrobacterium tumefaciens strain pGV3850 using standardtri-parental mating techniques with a helper plasmid pGJ23. Transformerswere selected using appropriate antibiotics. Total DNA was isolated fromselected strains, digested with several endonucleases, separated onagarose gels by electrophoresis, blotted to membrane and hybridized tovarious probes to confirm the presence of all parts of the respectivechimeric gene in Agrobacterium.

EXAMPLE SEVEN Creation of Transgenic Plants

Cotyledons from eight day old tomato plants were cut and placed ontobacco cell feeder layers for 24 hours before infecting with anAgrobacterium strain (as prepared according to Example 6) harboringplasmid containing a chimeric gene. After a 30 minute infection period,the cotyledons were placed back on the tobacco cell feeder layers andincubated for an additional 48 hours before transferring onto a tomatoregeneration medium containing kanamycin and cefotaxime. Every two weeksthe explants were subcultured into new regeneration media. Regeneratedshoots were rooted using a tomato rooting medium. The rooted plants wereremoved from tissue culture and placed in a growth chamber in soil for 2weeks. The plants were then moved to the greenhouse. The presence of theinserted DNA in transgenic plants was confirmed using DNA gel blottingof genomic DNA obtained from transformed (regenerated) tomato plants.

EXAMPLE EIGHT Level of FRS-LOX mRNA and Protein in Plants Transformedwith pMLSL

The levels of FRS-LOX mRNA and protein in ripening fruits from theprimary transgenic tomato plants expressing pMLSL analyzed were about50% of that wild type parental fruits. FRS-LOX mRNA and protein levelswere determined using RNA gel blotting and immuno blotting,respectively. Since seeds from these primary transgenic plantssegregated in a normal Mendelian manner they are believed to beherterozygous for the introduced pMLSL construct.

EXAMPLE NINE Effect of Reduced FRS-LOX on Ripening-Associated FruitFirmness

The level of firmness of whole transgenic fruits transformed with thenucleotide sequence of SEQ ID NO:1 in sense orientation were determinedby using a McCormick Fruit Tech (Yakima, Wash.) firmness pressuretester. This tester has a plunger tip and measures the penetration forcenecessary for the plunger tip to enter the fruit epidermis. Inperforming these tests, about a 1 cm square section of the waxyepidermis from each fruit was removed where the measurement was to betaken. A minimum of six fruits were analyzed and the mean and standarderror were calculated. FIG. 6 shows the comparison ofripening-associated fruit firmness over time post breaker in PMSL-650-1fruits (transgenic tomato transformed with the PMLSL vector) and wildtype parental plants. Table 3 shows the firmness of fruits fromsegregating progeny of PMSL-650-1 with zero, one and two copies of theinserted pMLSL gene. These data clearly demonstrate that reduction ofthe FRS-LOX inhibits ripening-associated fruit softening.

TABLE 3 Firmness of Fruits from Transgenic Tomato Plant Containing 0, 1and 2 copies of inserted pMLSL Gene. Days After Fruits Firmness, lbs.Breaker Copies of Inserted Gene Stage 0 1 2 7 8.85+/−0.96 9.51+/−0.6715.51+/−1.94 10 6.29+/−0.49 7.43+/−0.42 11.46+/−2.93

While the invention has been described in detail in the foregoingdescription, the same is considered as illustrative and not restrictivein character, it being understood that only the preferred embodimentshave been shown and described and that all changes and modificationsthat come within the spirit of the invention are desired to beprotected.

4 1 2441 DNA Lycopersicon esculentum 1 ttcagttgtt gatggcattt ctgatttacttggccaaaaa gtctctatcc 50 aattgataag tggttctgtt aattatgatg gtttggaagggaaactgagc 100 aatccagcat acttagagag ttggcttaca gacatcaccc caataacagc150 aggggaatca acttttagtg ttacatttga ctgggatcgt gacgagtttg 200gagttccagg agcattcatc atcaagaatc ttcatcttaa tgagttcttt 250 ctcaagtcactcacactcga agatgttcct aattatggaa aaatccattt 300 tgtatgcaat tcttgggtttatcctgcttt tagatacaag tctgaccgca 350 ttttctttgc caatcaggct tatctcccaagtgaaacacc acaaccattg 400 cgaaaataca gagaaaatga actggtagct ttgcgaggagatggaactgg 450 aaagcttgaa gaatgggaca gggtttatga ttatgcttgc tacaatgact500 tgggtgaacc agataagggg gaagagtatg ctaggcctat ccttggaggg 550tcctctgagt acccgtatcc tcgtagaggc aggacaggcc gcgaaccaac 600 caaagcagatcctaattgcg agagcaggaa cccattgcct atgagcttag 650 acatatatgt cccaagggacgagcgatttg gtcatgtgaa gaagtcagac 700 tttttgacgt cgtccttaaa atcctctttgcaaacgcttc tccctgcgtt 750 taaggctttg tgcgataaca cgcctaatga gttcaatagctttgcggatg 800 tacttaatct ctatgaagga ggaatcaagt tgcctgaagg cccttggttg850 aaagccatta ctgataacat ttcctcagag atactaaaag acatccttca 900aacggatggt caaggcctac ttaagtaccc aactcctcag gttattcaag 950 gcgataaaactgcatggagg acggatgaag aatttgggag agaaatgttg 1000 gcaggatcca atcctgtcttaatcagtaga ctccaagaat ttcctccgaa 1050 gagcaagttg gatccaacca tatatggaaaccaaaacagt acaattacca 1100 cagaacatgt acaggataag ttgaatggat taacagtgaatgaggcaatc 1150 aagagtaaca ggttattcat attgaaccac catgacatcg tgatgccact1200 attgaggaaa attaacatgt cagcaaacac aaaagcctat gcctcaagaa 1250ctctgctctt cctacaagat gatagaactt tgaagccact agcaattgaa 1300 ctaagcttgccacatccaga cggagatcaa tttggtactg ttagtaaagt 1350 atatacacca gctgaccaaggtgttgaagg ttctatctgg cagtttgcca 1400 aagcctatgt agcagtgaat gacatgggcattcatcagct cattagccac 1450 tggttgaata cacacgcggt gatcgaacca tttgtgattgcaacaaatag 1500 gcatctaagt gtgcttcatc ccattcataa acttcttcat cctcatttcc1550 gtaacacgat gaacataaat gctttagcaa gagagacctt gacctatgat 1600ggtggttttg agacgtctct ttttcctgcc aaatattcca tggaaatgtc 1650 agcagcagcttacaaagatt gggttttccc tgaacaagca cttcctgctg 1700 atctcctcaa aagaggagtggctgttgagg acttgagctc cccacatggc 1750 attcgtttac tgattctgga ctatccatatgctgttgatg gcttggaaat 1800 ttgggcagca atcaaaagtt gggtaacaga atattgcaagttctattaca 1850 aatctgacga gacagtagag aaagacactg aactccaagc ttggtggaag1900 gagctccgcg aagaaggaca tggcgacaag aaagatgagg cttggtggcc 1950taaactgcaa actcgacaag agctcagaga ttgttgcacc atcattatat 2000 ggatagcttcagcacttcat gcagcactcc attttggctt atactcttac 2050 gctggttatc tccctaatcgccctacttta agctgtaatt tgatgccaga 2100 gccaggaagt gttgagtatg aagagctcaagacaaatcca gacaaggtat 2150 tcctaaaaac atttgttcct cagttgcaat cactgcttgaaatttccatc 2200 tttgaggtct cgtcaaggca tgcttcagat gaggtttact tgggacaaag2250 ggactcaatt gaatggacaa aggataaaga accacttgta gcttttgaga 2300ggtttggaaa gatgctaagt gatatcgaga atcgaattat gataatgaat 2350 agtcataagagttggaagaa caggtcaggg cctgttaacg ttccatatac 2400 gttgctcttt cccacaagtgaagagggact cacaggcaaa g 2441 2 2871 DNA Lycopersicon esculentum 2ttttcttaat taaaaaaaaa atatttctgt ttaaatagtt aatcatgtct 50 ttgggtggaattgtggatgc catccttgga aaagatgata ggccaaaagt 100 gaaaggaaga gtgattttgatgaaaaaaaa tgttctagac ttcattaata 150 taggtgcttc agttgttgat ggcatttctgatttacttgg ccaaaaagtc 200 tctatccaat tgataagtgg ttctgttaat tatgatggtttggaagggaa 250 actgagcaat ccagcatact tagagagttg gcttacagac atcaccccaa300 taacagcagg ggaatcaact tttagtgtta catttgactg ggatcgtgac 350gagtttggag ttccaggagc attcatcatc aagaatcttc atcttaatga 400 gttctttctcaagtcactca cactcgaaga tgttcctaat tatggaaaaa 450 tccattttgt atgcaattcttgggtttatc ctgcttttag atacaagtct 500 gaccgcattt tctttgccaa tcaggcttatctcccaagtg aaacaccaca 550 accattgcga aaatacagag aaaatgaact ggtagctttgcgaggagatg 600 gaactggaaa gcttgaagaa tgggacaggg tttatgatta tgcttgctac650 aatgacttgg gtgaaccaga taagggggaa gagtatgcta ggcctatcct 700tggagggtcc tctgagtacc cgtatcctcg tagaggcagg acaggccgcg 750 aaccaaccaaagcagatcct aattgcgaga gcaggaaccc attgcctatg 800 agcttagaca tatatgtcccaagggacgag cgatttggtc atgtgaagaa 850 gtcagacttt ttgacgtcgt ccttaaaatcctctttgcaa acgcttctcc 900 ctgcgtttaa ggctttgtgc gataacacgc ctaatgagttcaatagcttt 950 gcggatgtac ttaatctcta tgaaggagga atcaagttgc ctgaaggccc1000 ttggttgaaa gccattactg ataacatttc ctcagagata ctaaaagaca 1050tccttcaaac ggatggtcaa ggcctactta agtacccaac tcctcaggtt 1100 attcaaggcgataaaactgc atggaggacg gatgaagaat ttgggagaga 1150 aatgttggca ggatccaatcctgtcttaat cagtagactc caagaatttc 1200 ctccgaagag caagttggat ccaaccatatatggaaacca aaacagtaca 1250 attaccacag aacatgtaca ggataagttg aatggattaacagtgaatga 1300 ggcaatcaag agtaacaggt tattcatatt gaaccaccat gacatcgtga1350 tgccactatt gaggaaaatt aacatgtcag caaacacaaa agcctatgcc 1400tcaagaactc tgctcttcct acaagatgat agaactttga agccactagc 1450 aattgaactaagcttgccac atccagacgg agatcaattt ggtactgtta 1500 gtaaagtata tacaccagctgaccaaggtg ttgaaggttc tatctggcag 1550 tttgccaaag cctatgtagc agtgaatgacatgggcattc atcagctcat 1600 tagccactgg ttgaatacac acgcggtgat cgaaccatttgtgattgcaa 1650 caaataggca tctaagtgtg cttcatccca ttcataaact tcttcatcct1700 catttccgta acacgatgaa cataaatgct ttagcaagag agaccttgac 1750ctatgatggt ggttttgaga cgtctctttt tcctgccaaa tattccatgg 1800 aaatgtcagcagcagcttac aaagattggg ttttccctga acaagcactt 1850 cctgctgatc tcctcaaaagaggagtggct gttgaggact tgagctcccc 1900 acatggcatt cgtttactga ttctggactatccatatgct gttgatggct 1950 tggaaatttg ggcagcaatc aaaagttggg taacagaatattgcaagttc 2000 tattacaaat ctgacgagac agtagagaaa gacactgaac tccaagcttg2050 gtggaaggag ctccgcgaag aaggacatgg cgacaagaaa gatgaggctt 2100ggtggcctaa actgcaaact cgacaagagc tcagagattg ttgcaccatc 2150 attatatggatagcttcagc acttcatgca gcactccatt ttggcttata 2200 ctcttacgct ggttatctccctaatcgccc tactttaagc tgtaatttga 2250 tgccagagcc aggaagtgtt gagtatgaagagctcaagac aaatccagac 2300 aaggtattcc taaaaacatt tgttcctcag ttgcaatcactgcttgaaat 2350 ttccatcttt gaggtctcgt caaggcatgc ttcagatgag gtttacttgg2400 gacaaaggga ctcaattgaa tggacaaagg ataaagaacc acttgtagct 2450tttgagaggt ttggaaagat gctaagtgat atcgagaatc gaattatgat 2500 aatgaatagtcataagagtt ggaagaacag gtcagggcct gttaacgttc 2550 catatacgtt gctctttcccacaagtgaag agggactcac aggcaaagga 2600 attcccaaca gtgtgtctat atagaacttattattcaatc agtttgttgt 2650 gcttgtgtta cttgttattc ccaaccaaat aaactctttgttccaaataa 2700 agagtattgt attgtattgt cttgtgtgtt gtgttgtatt gtattatatt2750 gtatagtatt attgatttaa atacaatgtt tgttgcactt gtttcttgtt 2800attcccaacc aaataaactc tttgttccaa ataaagctgt agttggtttt 2850 aaaaaaaaaaaaaaaaaaaa a 2871 3 297 DNA Lycopersicon esculentum 3 ttttcttaattaaaaaaaaa atatttctgt ttaaatagtt aatcatgtct 50 ttgggtggaa ttgtggatgccatccttgga aaagatgata ggccaaaagt 100 gaaaggaaga gtgattttga tgaaaaaaaatgttctagac ttcattaata 150 taggtgcttc agttgttgat ggcatttctg atttacttggccaaaaagtc 200 tctatccaat tgataagtgg ttctgttaat tatgatggtt tggaagggaa250 actgagcaat ccagcatact tagagagttg gcttacagac atcaccc 297 4 2871 DNALycopersicon esculentum 4 ttttcttaat taaaaaaaaa atatttctgt ttaaatagttaatcatgtct 50 ttgggtggaa ttgtggatgc catccttgga aaagatgata ggccaaaagt 100gaaaggaaga gtgattttga tgaaaaaaaa tgttctagac ttcattaata 150 taggtgcttcagttgttgat ggcatttctg atttacttgg ccaaaaagtc 200 tctatccaat tgataagtggttctgttaat tatgatggtt tggaagggaa 250 actgagcaat ccagcatact tagagagttggcttacagac atcaccccaa 300 taacagcagg ggaatcaact tttagtgtta catttgactgggatcgtgac 350 gagtttggag ttccaggagc attcatcatc aagaatcttc atcttaatga400 gttctttctc aagtcactca cactcgaaga tgttcctaat tatggaaaaa 450tccattttgt atgcaattct tgggtttatc ctgcttttag atacaagtct 500 gaccgcattttctttgccaa tcaggcttat ctcccaagtg aaacaccaca 550 accattgcga aaatacagagaaaatgaact ggtagctttg cgaggagatg 600 gaactggaaa gcttgaagaa tgggacagggtttatgatta tgcttgctac 650 aatgacttgg gtgaaccaga taagggggaa gagtatgctaggcctatcct 700 tggagggtcc tctgagtacc cgtatcctcg tagaggcagg acaggccgcg750 aaccaaccaa agcagatcct aattgcgaga gcaggaaccc attgcctatg 800agcttagaca tatatgtccc aagggacgag cgatttggtc atgtgaagaa 850 gtcagactttttgacgtcgt ccttaaaatc ctctttgcaa acgcttctcc 900 ctgcgtttaa ggctttgtgcgataacacgc ctaatgagtt caatagcttt 950 gcggatgtac ttaatctcta tgaaggaggaatcaagttgc ctgaaggccc 1000 ttggttgaaa gccattactg ataacatttc ctcagagatactaaaagaca 1050 tccttcaaac ggatggtcaa ggcctactta agtacccaac tcctcaggtt1100 attcaaggcg ataaaactgc atggaggacg gatgaagaat ttgggagaga 1150aatgttggca ggatccaatc ctgtcttaat cagtagactc caagaatttc 1200 ctccgaagagcaagttggat ccaaccatat atggaaacca aaacagtaca 1250 attaccacag aacatgtacaggataagttg aatggattaa cagtgaatga 1300 ggcaatcaag agtaacaggt tattcatattgaaccaccat gacatcgtga 1350 tgccactatt gaggaaaatt aacatgtcag caaacacaaaagcctatgcc 1400 tcaagaactc tgctcttcct acaagatgat agaactttga agccactagc1450 aattgaacta agcttgccac atccagacgg agatcaattt ggtactgtta 1500gtaaagtata tacaccagct gaccaaggtg ttgaaggttc tatctggcag 1550 tttgccaaagcctatgtagc agtgaatgac atgggcattc atcagctcat 1600 tagccactgg ttgaatacacacgcggtgat cgaaccattt gtgattgcaa 1650 caaataggca tctaagtgtg cttcatcccattcataaact tcttcatcct 1700 catttccgta acacgatgaa cataaatgct ttagcaagagagaccttgac 1750 ctatgatggt ggttttgaga cgtctctttt tcctgccaaa tattccatgg1800 aaatgtcagc agcagcttac aaagattggg ttttccctga acaagcactt 1850cctgctgatc tcctcaaaag aggagtggct gttgaggact tgagctcccc 1900 acatggcattcgtttactga ttctggacta tccatatgct gttgatggct 1950 tggaaatttg ggcagcaatcaaaagttggg taacagaata ttgcaagttc 2000 tattacaaat ctgacgagac agtagagaaagacactgaac tccaagcttg 2050 gtggaaggag ctccgcgaag aaggacatgg cgacaagaaagatgaggctt 2100 ggtggcctaa actgcaaact cgacaagagc tcagagattg ttgcaccatc2150 attatatgga tagcttcagc acttcatgca gcactccatt ttggcttata 2200ctcttacgct ggttatctcc ctaatcgccc tactttaagc tgtaatttga 2250 tgccagagccaggaagtgtt gagtatgaag agctcaagac aaatccagac 2300 aaggtattcc taaaaacatttgttcctcag ttgcaatcac tgcttgaaat 2350 ttccatcttt gaggtctcgt caaggcatgcttcagatgag gtttacttgg 2400 gacaaaggga ctcaattgaa tggacaaagg ataaagaaccacttgtagct 2450 tttgagaggt ttggaaagat gctaagtgat atcgagaatc gaattatgat2500 aatgaatagt cataagagtt ggaagaacag gtcagggcct gttaacgttc 2550catatacgtt gctctttccc acaagtgaag agggactcac aggcaaagga 2600 attcccaacagtgtgtctat atagaactta ttattcaatc agtttgttgt 2650 gcttgtgtta cttgttattcccaaccaaat aaactctttg ttccaaataa 2700 agagtattgt attgtattgt cttgtgtgttgtgttgtatt gtattatatt 2750 gtatagtatt attgatttaa atacaatgtt tgttgcacttgtttcttgtt 2800 attcccaacc aaataaactc tttgttccaa ataaagctgt agttggtttt2850 aaaaaaaaaa aaaaaaaaaa a 2871

What is claimed is:
 1. A DNA construct selected from the groupconsisting of pMLSL, pMLAL, pUSL2, pUAL2, pUEL300S and pUEL300A.
 2. Anisolated polynucleotide having a sequence selected from the groupconsisting of the sequence of SEQ ID NO:1, the sequence of SEQ ID NO:2,the sequence of SEQ ID NO:3, and the sequence of SEQ ID NO:4.
 3. A DNAconstruct causing a decrease in production of fruit ripening specificlipoxygenase in a plant cell transformed with the construct, theconstruct comprising an isolated polynucleotide having a sequenceselected from the group consisting of the sequence of SEQ ID NO: 1, thesequence of SEQ ID NO:2, the sequence of SEQ ID NO:3, and the sequenceof SEQ ID NO:4, wherein the polynucleotide is operably linked to atleast one regulatory sequence in either sense or antisense orientationwith respect to the regulatory sequence.
 4. The DNA construct accordingto claim 3, wherein the polynucleotide comprises the sequence set forthin SEQ ID NO:1.
 5. The DNA construct according to claim 3, wherein thepolynucleotide comprises the sequence set forth in SEQ ID NO:2.
 6. TheDNA construct according to claim 3, wherein the polynucleotide comprisesthe sequence set forth in SEQ ID NO:3.
 7. The DNA construct according toclaim 3, wherein the polynucleotide comprises the sequence set forth inSEQ ID NO:4.
 8. A transformed plant featuring a decreased production offruit ripening specific lipoxygenase relative to an untransformed plant,comprising a host plant having incorporated therein a DNA constructcausing a decrease in production of fruit ripening specificlipoxygenase, the construct comprising an isolated polynucleotide havinga sequence selected from the group consisting of the sequence of SEQ IDNO:1, the sequence of SEQ ID NO:2, the sequence of SEQ ID NO:3, and thesequence of SEQ ID NO:4, wherein the polynucleotide is operably linkedto at least one regulatory sequence in either sense or antisenseorientation with respect to the regulatory sequence.
 9. The transformedplant of claim 8 wherein the host plant is a tomato plant.
 10. A methodof altering the production of fruit ripening specific lipoxygenase in aplant, comprising stably incorporating into the genome of the plant aDNA construct causing a decrease in production of fruit ripeningspecific lipoxygenase in a plant cell transformed with the construct,the construct comprising a polynucleotide operably linked to at leastone regulatory sequence in either sense or antisense orientation withrespect to the regulatory sequence; wherein the polynucleotide has asequence selected from the group consisting of the sequence of SEQ IDNO:1, the sequence of SEQ ID NO:2, the sequence of SEQ ID NO:3, and thesequence of SEQ ID NO:4; and wherein said incorporating is achieved bytransformation means whereby the incorporated construct causes adecrease in production of fruit ripening specific lipoxygenase in aplant cell containing the polynucleotide as compared to that of anuntransformed plant.
 11. The method according to claim 10, wherein thepolynucleotide comprises the sequence set forth in SEQ ID NO:1.
 12. Themethod according to claim 10, wherein the polynucleotide comprises thesequence set forth in SEQ ID NO:2.
 13. The method according to claim 10,wherein the polynucleotide comprises the sequence set forth in SEQ IDNO:3.
 14. The method according to claim 10, wherein the polynucleotidecomprises the sequence set forth in SEQ ID NO:4.